Fluorescent 3-hydroxy-4-pyridinone hexadentate iron chelators: intracellular distribution and the relevance to antimycobacterial properties

Abstract

We report the synthesis and characterization of a fluorescent iron chelator (4), shown to be effective in inhibiting the growth of Mycobacterium avium in macrophages, together with the synthesis and characterization of two unsuccessful analogues selected to facilitate identification of the molecular properties responsible for the antimicrobial activity. Partition of the chelators in liposomes was investigated and the compounds were assessed with respect to uptake by macrophages, responsiveness to iron overload/iron deprivation and intracellular distribution by flow cytometry and confocal microscopy. The synthesis of the hexadentate chelators is based on a tetrahedral structure to which three bidentate 3-hydroxy-4-pyridinone chelating units are linked via amide bonds. The structure is synthetically versatile, allowing further addition of functional groups such as fluorophores. Here, we analyse the non-functionalized hexadentate unit (3) and the corresponding rhodamine B (4) and fluorescein (5) labelled chelators. The iron(III) stability constant was determined for 3 and the values log beta = 34.4 and pFe(3+) = 29.8 indicate an affinity for iron of the same order of magnitude as that of mycobacteria siderophores. Fluorescence properties in the presence of liposomes show that 4 strongly interacts with the lipid phase, whereas 5 does not. Such different behaviour may explain their distinct intracellular localization as revealed by confocal microscopy. The flow cytometry and confocal microscopy studies indicate that 4 is readily engulfed by macrophages and targeted to cytosol and vesicles of the endolysosomal continuum, whereas 5 is differentially distributed and only partially colocalizes with 4 after prolonged incubation. Differential distribution of the compounds is likely to account for their different efficacy against mycobacteria.

A Novel Antimycobacterial Compound Acts as an Intracellular Iron Chelator

“…Chelators are able to restrict growth of Mycobacterium avium (46)(47)(48), while synthetic mycobactin analogues selectively inhibit M. tuberculosis growth (49). Additionally, iron chelation has proved to inhibit the cytotoxic effect of mycolactone, a macrolide toxin produced by Mycobacterium ulcerans, the causative agent of the necrotizing skin disease Buruli ulcer (50).…”

Synthesis of novel Iron(III) chelators based on triaza macrocycle backbone and 1-hydroxy-2(H)-pyridin-2-one coordinating groups and their evaluation as antimicrobial agents

“…A significant number of reports have also described growth inhibition of a range of pathogenic bacteria by bidentate or hexadentate chelators based upon 3.4-HOPO [34][35][36][37][38][39][40][41][42][43]. To achieve high pFe 3+ , hexadentate chelators are preferred to bidentate ones, especially since their mode of action as biostatic agents is expected to be extracellular and therefore do not suffer from size restriction to penetrate the microbes.…”

Iron speciation in natural waters by sequential injection analysis with a hexadentate 3-hydroxy-4-pyridinone chelator as chromogenic agent

“…Step previously described by one of us [4]. The values of log β(Fe 3 þ ) and pFe 3 þ values are 34.4 and 29.8, respectively, and formation of the complex is fully achieved at pH above 3.…”

“…The hexadentate 3,4-hydroxypiridinone ligand (a tripodal hydroxypyridinone) was synthesised according to the methods published in the literature [4,5] and characterized by elemental analysis, 1 H and 13 C NMR and mass spectrometry. Ligand solutions were obtained by dissolution of approximately 10.0 mg of the synthesised ligand in 20.0 mL of water corresponding to a concentration of 0.5 mg/L (521 mM).…”

“…So, to tackle this limitation, a more soluble and hexadentate 3-hydroxy-4-pyridinone ligand ( Fig. 1) [4][5][6] was explored in this work.…”